Translatome proteomics identifies autophagy as a resistance mechanism to on-target FLT3 inhibitors in acute myeloid leukemia

Internal tandem duplications (ITD) in the receptor tyrosine kinase FLT3 occur in 25 % of acute myeloid leukemia (AML) patients, drive leukemia progression and confer a poor prognosis. Primary resistance to FLT3 kinase inhibitors (FLT3i) quizartinib, crenolanib and gilteritinib is a frequent clinical challenge and occurs in the absence of identifiable genetic causes. This suggests that adaptive cellular mechanisms mediate primary resistance to on-target FLT3i therapy. Here, we systematically investigated acute cellular responses to on-target therapy with multiple FLT3i in FLT3-ITD + AML using recently developed functional translatome proteomics (measuring changes in the nascent proteome) with phosphoproteomics. This pinpointed AKT-mTORC1-ULK1-dependent autophagy as a dominant resistance mechanism to on-target FLT3i therapy. FLT3i induced autophagy in a concentration- and time-dependent manner specifically in FLT3-ITD + cells in vitro and in primary human AML cells ex vivo. Pharmacological or genetic inhibition of autophagy increased the sensitivity to FLT3-targeted therapy in cell lines, patient-derived xenografts and primary AML cells ex vivo. In mice xenografted with FLT3-ITD + AML cells, co-treatment with oral FLT3 and autophagy inhibitors synergistically impaired leukemia progression and extended overall survival. Our findings identify a molecular mechanism responsible for primary FLT3i treatment resistance and demonstrate the pre-clinical efficacy of a rational combination treatment strategy targeting both FLT3 and autophagy induction.

proteomics, 1 mg of pooled peptides were used for phosphopeptide enrichment by High-Select Fe-NTA Phosphopeptide enrichment kit (Thermo Fisher Scientific) following the manufacturer's instructions.
After enrichment, peptides were dried and resuspended in 70 % acetonitrile/0.1 % TFA and filtered through a C8 (Empore, Thermo Fisher Scientific) stage tip to remove contaminating Fe-NTA particles.
Dried phosphopeptides were then fractionated on a C18 (Empore, Thermo Fisher Scientific) stage tip.
For fractionation, C18 stage tips were washed with 100 % acetonitrile twice, followed by equilibration

Offline high-pH reverse phase fractionation
Peptides were fractionated using a Dionex Ultimate 3000 analytical HPLC. 240 µg of pooled and purified TMT-labeled samples were resuspended in 10 mM ammonium bicarbonate (ABC), 5 % acetonitrile (ACN) and separated on a 250 mm long C18 column (Waters XBridge C18, 4.6 mm x 250 mm, 3.5 µm particle size) using a multistep gradient from 100 % solvent A (5 % AC, 10 mM ABC in water) to 60 % solvent B (90 % CAN, 10 mM ABC in water) over 70 min. Eluting peptides were collected every 45 s for a total of 96 fractions, which were then cross-concatenated into 24 fractions and dried for further processing.

Liquid chromatography mass spectrometry
For translatome analysis, mass spectrometry data were acquired in centroid mode on a QExactive HF mass spectrometer hyphenated to an Easy nLC 1200 nano HPLC system using a nanoFlex ion source (Thermo Fisher Scientific) applying a spray voltage of 2.3 kV. Peptides were separated on a self-made, 35 cm long, 75 µm ID fused-silica column, packed in-house with 1.9 µM C18 particles (ReproSil-Pur, Dr. Maisch, Ammerbuch, Germany) and heated to 50 °C using an integrated column oven (Sonation, Biberach, Germany). HPLC solvents consisted of 0.1 % formic acid in water (buffer A) and 0.1 % formic acid, 80 % acetonitrile in water (buffer B). Each peptide fraction was eluted by a non-linear gradient from 5 %-30 % B over 90 min, followed by a stepwise increase to 95 % B in 6 min, which was held for another 9 min. Full scan MS spectra (350-1400 m/z) were acquired at a resolution of 120,000 at m/z 200, a maximum injection time of 100 ms and an automatic gain control (AGC) target value of 3x10 6 . The 20 most intense precursors per full scan with a charge state between 2 and 6 were isolated using a 1 Th window and fragmented using higher energy collisional dissociation (HCD, normalized collision energy (NCE) of 35%). MS/MS spectra were acquired with a resolution of 45,000 at m/z 200, a maximum injection time of 86 ms and an AGC target value of 1x10 5 . Dynamic exclusion was set to 20 s to limit repeated sequencing of previously acquired precursors.
For phosphopeptide analysis, mass spectrometry data were acquired in centroid mode on an Orbitrap Fusion Lumos mass spectrometer hyphenated to an Easy nLC 1200 nano HPLC system using a nanoFlex ion source (Thermo Fisher Scientific) applying a spray voltage of 2.6 kV with the transfer tube heated to 300 °C and a funnel RF of 30%. Internal mass calibration was enabled (lock mass 445.12003 m/z). Peptides were separated on a self-made, 35 cm long, 75 µm ID fused-silica column, packed in-house with 1.9 µM C18 particles (ReproSil-Pur, Dr. Maisch) and heated to 50 °C using an integrated column oven (Sonation). HPLC solvents consisted of 0.1 % formic acid in water (buffer A) and 0.1 % formic acid, 80 % acetonitrile in water (buffer B). Each peptide fraction was eluted by a nonlinear gradient from 3 %-35 % B over 120 min, followed by a stepwise increase to 90 % B in 6 min, which was held for another 9 min. Full scan MS spectra (350-1400 m/z) were acquired at a resolution of 120,000 at m/z 200, a maximum injection time of 100 ms and an AGC target value of 4x10 5 . The 20 most intense precursors per full scan with a charge state between 2 and 5 were selected for fragmentation, isolated with a quadrupole isolation window of 0.7 Th and fragmented via HCD, applying a NCE of 38 %. MS/MS were acquired in the Orbitrap with a resolution of 50,000 at m/z 200, a maximum injection time of 86 ms and an AGC target value of 1x10 5 . Dynamic exclusion was set to 60 s and 7 ppm to limit repeated sequencing of already acquired precursors and advanced peak determination was deactivated.

Mass spectrometry data analysis
Raw files were analyzed using Proteome Discoverer (PD) 2.4 (Thermo Fisher Scientific). Spectra were selected using default settings. For total proteome/translatome analysis, database searches were performed using SequestHT node in PD against trypsin-digested Homo sapiens SwissProt database +42.0.11) and phosphorylation (Ser/Thr/Tyr, +79.966). After search, identifications were validated using a concatenated target-decoy strategy and FDR was estimated using q-values calculated by Percolator, applying 1 % and 5 % cut-offs for high and medium confidence hits. Only high-confidence hits were accepted for further analysis. Phosphosite localization probabilities were calculated using the ptmRS node in PhosphoRS mode and default settings. Consensus workflow for reporter ion quantification was performed with default settings, except the minimal signal-to-noise ratio was set to 5 for translatome analysis. Results were exported as Excel files for further processing.
Excel files were used as input for a custom-made in-house R pipeline. R version 4.0.2 was used together with data.table 1.13.0. Excel files with PSM data were read in and sample loading normalization was performed by total intensity normalization to the lowest channel. For mePROD analysis of translation, all possible modification states/PSMs containing a heavy label were extracted. Baseline subtraction was performed on PSM level by subtracting the measured intensities of the non-SILAC-labeled sample from all other channel values (1). Negative intensities were treated as zero. PSM data were then collapsed onto protein-level by intensity summation of PSMs belonging to the same unique protein. Internal reference scaling (IRS) (2) to one physically identical bridge channel included in all TMT multiplexes was applied to correct for LC/MS sampling batch effects. For this, intensity values were geometricaveraged across the bridge channels from individual multiplexes for each protein, the IRS normalization factor for each multiplex and protein was calculated by dividing this target value by the bridge channel intensity value, and individual protein intensities were scaled by multiplication with each protein's IRS factor per multiplex.

RNA-Seq data analysis
Illumina paired-end sequencing run files obtained from NCBI Sequence Read Archive were pseudoaligned to the human reference transcriptome and reads were quantified using kallisto 0.46.1 (3) (100 bootstrap samples, default seed, sequence-based bias correction enabled). The transcriptome index for kallisto (k-mer default 31) was built from the human reference transcriptome GRCh38 (hg38) release 105 obtained from Ensembl. Differential expression analyses of transcripts and aggregation to the gene level was done with sleuth 0.30.0 (4), using the counts-aggregation method to obtain gene-level mean fold changes for pairwise contrasts and adjusted q-values from P values (likelihood ratio test) aggregated using lancaster method for gene-level pairwise contrasts, both as implemented in sleuth.

Generation of transgene cells
Lentivirus was generated by co-transfecting Lenti-X 293T cells with indicated vectors, psPAX2 and pMD2G plasmids using polyethylenimine. Medium was exchanged after 16 h and viral supernatant was harvested 72 hours after transfection, aliquoted and stored at −80 °C. Cells were transduced by spinfection (300 g for 1 h at 34 °C, 6.25 µg/ml polybrene). The next day, medium was exchanged and 72h after transduction, cells were selected by the addition of appropriate antibiotics (2 µg/ml puromycin; 8 µg/ml blasticidin; 300 µg/ml hygromycin). Cells were not selected for single clones and experiments were performed on bulk cell populations. 32Dcl3 cells were retrovirally transduced with human FLT3-ITD (12 aa insertion between wildtype FLT3 aa595 and aa596) in pAULO vector (5). To generate autophagic flux reporter cells, cells were lentivirally transduced with eGFP-hLC3B 1-120 -mCherry-P2Apuromycin or GFP-LC3B-RFP-P2A-hygromycin at low multiplicity (MOI < 0.1). Transduction efficiency was measured by flow cytometry 72 h after transduction For targeted deletions, cells were further lentivirally transduced with a lentiCRISPRv2-based expression vector at MOI < 0.1 to express SpCas9-P2A-blasticidin, and then with lentiviral gRNA vectors. shRNA was similarly introduced by lentiviral transduction of parental cells and puromycin selection.
lentiCRISPRv2 was a gift from Feng Zhang (Addgene plasmid #52961). For stable expression of autophagy flux reporter, lentiCRISPRv2 was modified by removal of the U6 promoter, gRNA spacer and the SpCas9 coding sequence was replaced with EGFP-LC3B-mCherry using standard cloning techniques. For stable expression of autophagy flux reporter with Cas9, lentiCRISPRv2 was modified by removal of the U6 promoter, gRNA spacer, exchange of the SpCas9 coding sequence with GFP-LC3-RFP and replacement of the puromycin cassette with a hygromycin cassette. For stable SpCas9 expression, lentiCRISPRv2 was modified by removal of the U6 promoter, gRNA spacer and the puromycin resistance gene was replaced by blasticidin resistance gene. For lentiviral packaging pMD2.G (Addgene #12259) and psPAX2 (Addgene #12260) were used

Autophagy assay by confocal microscopy imaging: Image acquisition and analysis
Images were acquired at a resolution of 512 x 512 pixels and a z slice distance of 1 µm. Cytosolic LC3 signal intensity was integrated per cell. Quantification analysis of LC3 stains was performed using NIH ImageJ 2.0.0 (6) and Bio-Formats plugin (7). At least 30-100 cells per experimental condition and replicate were analyzed and counted. All individual z sections of images with multiple cells were analyzed to obtain the total cytosolic LC3 signal intensity integrated per cell after image background subtraction and subtraction of nuclear, DAPI-overlapping signal.

Viability and proliferation assays
Cells were seeded into white 96-well plates ( Harbour, ME, USA), isolated from bone marrow or spleen at advanced leukemia as described previously (9) and cryopreserved. After thawing, cells were cultured for 42 h prior to treatments as described for primary AML cells, except for additional supplementation of 1% penicillin/streptomycin and 10 ng/ml human thrombopoetin (STEMCELL Technologies). Firefly luciferase expression was measured using ONE-Glo EX (Promega) following the manufacturer's recommendations.

Flow cytometry
All samples were analyzed on a LSRFortessa cytometer (BD) or a FACSCelesta (BD) equipped with a HTS unit. Measurements and analyses were performed using FACSDiva software 7.0 (BD) and FlowJo software 10.7.1 (Tree Star). Instrument setup including fluorescence amplification and compensation was performed by applying FACSDiva compensation setup. Flow cytometer performance was checked regularly using CS&T beads (BD).  (10)